The objective of this proposed research is to define the role of mitochondrial leucyl-tRNA synthetase and elongation factor TU (EFTU) in the pathogenesis of mutations in the tRNALeu (UUR) that are commonly related to human diseases. This second goal of this study is to explore a way to therapeutically intervene for maternally inherited disease by over-expression of these two proteins in the cybrids carrying pathogenic mutations in the tRNA Leu (UUR). A variety of mitochondrial DNA (mtDNA) mutation have been found to be associated with many clinical abnormalities, including neuromuscular disorders. Of the 100 pathogenic point mutations in mtDNA, >70 occur in tRNA genes. Of these, the most common mutation is the A to G transition at position 3243 (A3243G) in the tRNALeu (UUR) gene, which causes mitochondrial encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS) and other disorders. Mitochondria with this mutated tRNA exhibited a reduced ATP production, which results from quantitative deficiencies in mitochondrial protein synthesis. We hypothesize that the over-expression of human mitochondrial leucyl-tRNA synthetase and EFTU in the disease cell model of the A3243G mutation in the tRNALeu (UUR) gene will correct the mitochondrial translational defects, consequently increasing the level of ATP production. This application proposes two specific aims: 1). Construction of the stably transfected cell lines through transferring human mitochondrial leucyl-tRNA synthetase and EFTU cDNAs into the cybrid cell lines carrying the A3243G mutation and wild type mtDNA. 2). These stably transfected cell lines will be evaluated for the correction of mitochondrial dysfunction by using biochemical and metabolic assays. Success of this protection will define the role of the mitochondrial leucyl-tRNA synthetase and EFTU in the pathogenesis of mutations in the tRNALeu(UUR) gene associated with human diseases. This should provide new insights into the molecular mechanism of maternally inherited disorders. In particular, success in the aim of correcting the biochemical defect associated with A3243G mutation in the tRNA Leu (UUR) gene by overexpressing mitochondrial leucyl-tRNA synthetase, will open the way to therapeutic interventions for maternally inherited diseases. In the long term, fundamental experimental approaches and knowledge, which results from this proposed work would be applicable to many other tRNA gene mutations, associated diseases in humans.